Process for Conversion of Granular Starch to Ethanol

ABSTRACT

The present invention concerns a method of producing glucose from a granular starch substrate comprising, contacting a slurry comprising granular starch obtained from plant material with an alpha-amylase at a temperature below the starch gelatinization temperature of the granular starch to produce oligosaccharides and hydrolyzing the oligosaccharides to produce a mash comprising at least 20% glucose and further comprising fermenting the mash to obtain ethanol.

FIELD OF THE INVENTION

The present invention relates to processes for the production of analcohol (e.g., ethanol) from a granular starch comprising exposing aslurry comprising granular starch from plant material to analpha-amylase at a temperature below the gelatinization temperature ofthe granular starch followed by fermentation with a fermentingmicroorganism.

BACKGROUND OF THE INVENTION

The commercial viability of producing ethanol as a fuel source fromagricultural crops has generated renewed worldwide interest due to avariety of reasons that include continued and increased dependence onlimited oil supplies and the fact that ethanol production is a renewableenergy source.

Alcohol fermentation production processes and particularly ethanolproduction processes are generally characterized as wet milling or drymilling processes. Reference is made to et al., 2005, Appl. Microbiol.Biotechnol. 67:19-25 and THE ALCOHOL TEXTBOOK, 3^(rd) Ed (K. A. Jacqueset al. Eds) 1999 Nottingham University Press, UK for a review of theseprocesses.

In general, the wet milling process involves a series of soaking(steeping) steps to soften the cereal grain wherein soluble starch isremoved followed by recovery of the germ, fiber (bran) and gluten(protein). The remaining starch is further processed by drying, chemicaland/or enzyme treatments. The starch may then be used for alcoholproduction, high fructose corn syrup or commercial pure grade starch.

In general, dry grain milling involves a number of basic steps, whichinclude: grinding, cooking, liquefaction, saccharification, fermentationand separation of liquid and solids to produce alcohol and otherco-products. Generally, whole cereal, such as corn cereal, is ground toa fine particle size and then mixed with liquid in a slurry tank. Theslurry is subjected to high temperatures in a jet cooker along withliquefying enzymes (e.g. alpha-amylases) to solubilize and hydrolyze thestarch in the cereal to dextrins. The mixture is cooled down and furthertreated with saccharifying enzymes (e.g. glucoamylases) to producefermentable glucose. The mash containing glucose is then fermented forapproximately 24 to 120 hours in the presence of ethanol producingmicroorganisms. The solids in the mash are separated from the liquidphase and ethanol and useful co-products such as distillers' grains areobtained.

Improvements to the above fermentation processes have been accomplishedby combining the saccharification step and fermentation step in aprocess referred to as simultaneous saccharification and fermentation orsimultaneous saccharification, yeast propagation and fermentation. Theseimproved fermentation processes have advantages over the previouslydescribed dry milling fermentation or even wet milling fermentationprocesses because significant sugar concentrations do not develop in thefermenter thereby avoiding sugar inhibition of yeast growth. Inaddition, bacterial growth is reduced due to lack of easily availableglucose. Increased ethanol production may result by use of thesimultaneous saccharification and fermentation processes.

More recently, fermentation processes have been introduced whicheliminate the cooking step or which reduce the need for treating cerealgrains at high temperatures. These fermentation processes which aresometimes referred to as no-cook, low temperature or warm cook, includemilling of a cereal grain and combining the ground cereal grain withliquid to form a slurry which is then mixed with one or more granularstarch hydrolyzing enzymes and optionally yeast at temperatures belowthe granular starch gelatinization temperature to produce ethanol andother co-products (U.S. Pat. No. 4,514,496, WO 03/066826; WO 04/081193;WO 04/106533; WO 04/080923 and WO 05/069840).

While the above mentioned fermentation processes using a milled grainslurry in combination with granular starch hydrolyzing enzymes offercertain improvements over previous processes, additional fermentationprocess improvements are needed by the industry for the conversion ofgranular starch resulting in higher energy efficiency and highend-product production. The object of the present invention is toprovide improved processes for the conversion of granular starch intoalcohol (e.g. ethanol) and other end products.

SUMMARY OF THE INVENTION

The present invention provides processes for producing an alcohol (e.g.ethanol) from granular starch by contacting the granular starch with analpha-amylase and providing suitable conditions for endogenous planthydrolytic enzymes, which hydrolyze solubilized starch to produceglucose. The glucose may then be used as a feedstock in fermentations toproduce alcohol.

In one aspect, the invention relates to a process of producing glucosefrom a granular starch substrate comprising:

-   -   a) contacting a slurry comprising granular starch obtained from        plant material with an alpha-amylase at a temperature below the        starch gelatinization temperature of the granular starch to        produce oligosaccharides and allowing endogenous plant        carbohydrate hydrolyzing enzymes to hydrolyze the        oligosaccharides, and    -   b) producing a mash comprising at least 10% glucose.

In a further embodiment of this aspect, the mash is fermented in thepresence of a fermenting microorganism and starch hydrolyzing enzymes ata temperature of between 10° C. and 40° C. for a period of time of 10hours to 250 hours to produce alcohol, particularly ethanol.

In another aspect, the invention relates to a process for producingethanol comprising:

-   -   a) contacting a slurry comprising granular starch with an        alpha-amylase capable of solubilizing granular starch, wherein        said contacting is at a pH of 3.5 to 7.0; at a temperature below        the starch gelatinization temperature of the granular starch;        and for a period of 5 minutes to 24 hours and obtaining a mash        substrate comprising greater than 20% glucose, and    -   b) fermenting the substrate in the presence of a fermenting        microorganism and a starch hydrolyzing enzyme at a temperature        of between 10° C. and 40° C. for a period of 10 hours to 250        hours to produce ethanol.

In further embodiments of either aspect described above, the processincludes recovering the ethanol. In yet further embodiments of thedescribed aspects, the process may include additional steps notspecified which are performed prior to, during or after the enumeratedsteps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a general schematic diagram that illustrates an embodiment ofthe invention wherein the slurry comprising a milled grain containinggranular starch and having a DS of 20 to 40% is contacted with analpha-amylase at a temperature between 55° C. to 70° C. and a pH of 4.0to 6.0 for 2 to 24 hours. The resulting mash comprising glucose istransferred to a fermentor and fermented at pH 3.0 to 5.0 at atemperature of 25° C. to 35° C. for 24 to 72 hours in the presence ofyeast, nutrients, acid and starch hydrolyzing enzymes to produceethanol.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are described.

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

DEFINITIONS

The term “fermentation” refers to the enzymatic and anaerobic breakdownof organic substances by microorganisms to produce simpler organiccompounds. While fermentation occurs under anaerobic conditions it isnot intended that the term be solely limited to strict anaerobicconditions, as fermentation also occurs in the presence of oxygen.

As used herein the term “starch” refers to any material comprised of thecomplex polysaccharide carbohydrates of plants, comprised of amylose andamylopectin with the formula (C₆H₁₀O₅)_(x), wherein x can be any number.

The term “granular starch” refers to raw (uncooked) starch, that isstarch in its natural form found in plant material (e.g. grains andtubers).

As used herein the term “dry solids content (DS)” refers to the totalsolids of a slurry in % on a dry weight basis.

The term “slurry” refers to an aqueous mixture comprising insolublesolids, (e.g. granular starch).

The term “dextrins” refers to short chain polymers of glucose (e.g. 2 to10 units).

The term “oligosaccharides” refers to any compound having 2 to 10monosaccharide units joined in glycosidic linkages. These short chainpolymers of simple sugars include dextrins.

The term “soluble starch” refers to starch which results from thehydrolysis of insoluble starch (e.g. granular starch).

The term “mash” refers to a mixture of a fermentable substrate in liquidused in the production of a fermented product and is used to refer toany stage of the fermentation from the initial mixing of the fermentablesubstrate with one or more starch hydrolyzing enzymes and fermentingorganisms through the completion of the fermentation run.

The terms “saccharifying enzyme” and “starch hydrolyzing enzymes” referto any enzyme that is capable of converting starch to mono- oroligosaccharides (e.g. a hexose or pentose).

The terms “granular starch hydrolyzing (GSH) enzyme” and “enzymes havinggranular starch hydrolyzing (GSH) activity” refer to enzymes, which havethe ability to hydrolyze starch in granular form.

The term “hydrolysis of starch” refers to the cleavage of glucosidicbonds with the addition of water molecules.

The term “alpha-amylase (e.g., E.C. class 3.2.1.1)” refers to enzymesthat catalyze the hydrolysis of alpha-1,4-glucosidic linkages. Theseenzymes have also been described as those effecting the exo orendohydrolysis of 1,4-α-D-glucosidic linkages in polysaccharidescontaining 1,4-α-linked D-glucose units.

The term “gelatinization” means solubilization of a starch molecule bycooking to form a viscous suspension.

The term “gelatinization temperature” refers to the lowest temperatureat which gelatinization of a starch containing substrate begins. Theexact temperature of gelatinization depends on the specific starch andmay vary depending on factors such as plant species and environmentaland growth conditions.

The term “below the gelatinization temperature” refers to a temperaturethat is less than the gelatinization temperature.

The term “glucoamylase” refers to the amyloglucosidase class of enzymes(e.g., E.C.3.2.1.3, glucoamylase, 1,4-alpha-D-glucan glucohydrolase).These are exo-acting enzymes, which release glucosyl residues from thenon-reducing ends of amylose and amylopectin molecules. The enzymes alsohydrolyzes alpha-1, 6 and alpha-1, 3 linkages although at much slowerrate than alpha-1, 4 linkages.

The phrase “simultaneous saccharification and fermentation (SSF)” refersto a process in the production of end products in which a fermentingorganism, such as an ethanol producing microorganism, and at least oneenzyme, such as a saccharifying enzyme are combined in the same processstep in the same vessel.

The term “saccharification” refers to enzymatic conversion of a directlyunusable polysaccharide to a mono- or oligosaccharide for fermentativeconversion to an end product.

The term “milling” refers to the breakdown of cereal grains to smallerparticles. In some embodiments the term is used interchangeably withgrinding.

The term “dry milling” refers to the milling of dry whole grain, whereinfractions of the grain such as the germ and bran have not been purposelyremoved.

The term “liquefaction” refers to the stage in starch conversion inwhich gelatinized starch is hydrolyzed to give low molecular weightsoluble dextrins.

The term “thin-stillage” refers to the resulting liquid portion of afermentation which contains dissolved material and suspended fineparticles and which is separated from the solid portion resulting fromthe fermentation. Recycled thin-stillage in industrial fermentationprocesses is frequently referred to as “back-set”.

The term “vessel” includes but is not limited to tanks, vats, bottles,flasks, bags, bioreactors and the like. In one embodiment, the termrefers to any receptacle suitable for conducting the saccharificationand/or fermentation processes encompassed by the invention.

The term “end product” refers to any carbon-source derived product whichis enzymatically converted from a fermentable substrate. In somepreferred embodiments, the end product is an alcohol, such as ethanol.

As used herein the term “fermenting organism” refers to anymicroorganism or cell which is suitable for use in fermentation fordirectly or indirectly producing an end product.

As used herein the term “ethanol producer” or ethanol producingmicroorganism” refers to a fermenting organism that is capable ofproducing ethanol from a mono- or oligosaccharide.

The term “derived” encompasses the terms “originated from”, “obtained”or “obtainable from”, and “isolated from” and in some embodiments asused herein means that a polypeptide encoded by the nucleotide sequenceis produced from a cell in which the nucleotide is naturally present orin which the nucleotide has been inserted.

The term “heterologous” with reference to a protein or polynucleotiderefers to a protein or polynucleotide that does not naturally occur in ahost cell.

The term “endogenous” with reference to a protein or polynucleotiderefers to a protein or polynucleotide that does naturally occur in ahost cell.

The phrase “endogenous plant hydrolytic enzymes capable of hydrolyzingsoluble starch” refers to hydrolytic enzymes that are expressed andproduced in a plant and may be produced by the expression of endogenousor heterologous genes.

The term “enzymatic conversion” in general refers to the modification ofa substrate by enzyme action. The term as used herein also refers to themodification of a fermentable substrate, such as a granular starchcontaining substrate by the action of an enzyme.

The terms “recovered”, “isolated”, and “separated” as used herein referto a compound, protein, cell, nucleic acid or amino acid that is removedfrom at least one component with which it is naturally associated.

As used herein the term “enzyme unit” refers to the amount of enzymethat produces 1 micromole of product per minute under the specifiedconditions of the assay. For example, in one embodiment, the term“glucoamylase activity unit” (GAU) is defined as the amount of enzymerequired to produce 1 g of glucose per hour from soluble starchsubstrate (4% DS) under assay conditions of 60° C. and pH 4.2.

The term “yield” refers to the amount of end product produced using themethods of the present invention. In some embodiments, the term refersto the volume of the end product and in other embodiments, the termrefers to the concentration of the end product.

The term “DE” or “dextrose equivalent” is an industry standard formeasuring the concentration of total reducing sugars, calculated asD-glucose on a dry weight basis. Unhydrolyzed granular starch has a DEthat is essentially 0 and D-glucose has a DE of 100. An instructivemethod for determining the DE of a slurry or solution is described inSchroorl's method (Fehling's assay titration).

As used herein the term “comprising” and its cognates are used in theirinclusive sense; that is, equivalent to the term “including” and itscorresponding cognates.

“A”, “an” and “the” include plural references unless the context clearlydictates otherwise.

Numeric ranges are inclusive of the numbers defining the range.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention, which can be had by reference to thespecification as a whole.

EMBODIMENTS OF THE INVENTION (A) Raw Materials Granular Starch—

Granular starch may be obtained from plant material including but notlimited to wheat, corn, rye, sorghum (milo), rice, millet, barley,triticale, cassaya (tapioca), potato, sweet potato, sugar beets,sugarcane, and legumes such as soybean and peas. Preferred plantmaterial includes corn, barley, wheat, rice, milo and combinationsthereof. Particularly preferred plant material is obtained from corn(Zea mays). Plant material may include hybrid varieties and geneticallymodified varieties (e.g. transgenic corn, barley or soybeans comprisingheterologous genes). Any part of the plant may be used to providegranular starch including but not limited to plant parts such as leaves,stems, hulls, husks, tubers, cobs, grains and the like. In someembodiments, essentially the entire plant may be used, for example, theentire corn stover may be used. In one embodiment, whole grain may beused as a source of granular starch. Preferred whole grains includecorn, wheat, rye, barley, sorghum and combinations thereof. In otherembodiments, granular starch may be obtained from fractionated cerealgrains including fiber, endosperm and/or germ components. Methods forfractionating plant material such as corn and wheat are known in theart. In some embodiments, plant material obtained from different sourcesmay be mixed together to obtain granular starch used in the processes ofthe invention (e.g. corn and milo or corn and barley).

In some embodiments, plant material comprising granular starch may beprepared by means such as milling. In particular, means of milling wholecereal grains are well known and include the use of hammer mills androller mills.

Alpha-Amylases—

In some of the embodiments encompassed by the invention, thealpha-amylase is a microbial enzyme having an E.C. number, E.C.3.2.1.1-3 and in particular E.C. 3.2.1.1. Any suitable alpha-amylase maybe used in the present process. In some embodiments, the alpha-amylaseis derived from a bacterial strain and in other embodiments thealpha-amylase is derived from a fungal strain. In further embodiments,the preferred alpha-amylase is a bacterial alpha-amylase. In otherembodiments, the alpha-amylase is an acid stable alpha-amylase. Suitablealpha-amylases may be naturally occurring as well as recombinant (hybridand variants) and mutant alpha-amylases (WO 99/19467 and WO 97/41213).In particularly preferred embodiments, the alpha-amylase is derived froma Bacillus species. Preferred Bacillus species include B. subtilis, B.stearothermophilus, B. lentus, B. licheniformis, B. coagulans, and B.amyloliquefaciens (U.S. Pat. No. 5,093,257; U.S. Pat. No. 5,763,385;U.S. Pat. No. 5,824,532; U.S. Pat. No. 5,958,739; U.S. Pat. No.6,008,026, U.S. Pat. No. 6,361,809; U.S. Pat. No. 6,867,031; WO96/23874; WO 96/39528 and WO 05/001064). Particularly preferredalpha-amylases are derived from Bacillus strains B. stearothermophilus,B. amyloliquefaciens and B. licheniformis ((U.S. Pat. No. 6,187,576;U.S. Pat. No. 6,093,562; U.S. Pat. No. 5,958,739; US 2006/0014265 and WO99/19467).

Most preferred alpha-amylases are amylases derived from B.stearothermophilus and B. licheniformis including wild-type, hybrid andvariant alpha-amylase enzymes. See Suzuki et al., (1989) J. Biol. Chem.264:18933-18938 and US 2006/0014265, particularly SEQ ID NOs: 3, 4 and16. Reference is also made to strains having American Type CultureCollection (ATCC) numbers—ATCC 39709; ATCC 11945; ATCC 6598; ATCC 6634;ATCC 8480; ATCC 9945A and NCIB 8059.

In addition to the bacterial alpha-amylases, fungal alpha-amylases arecontemplated for use in the processes of the invention. Suitable fungalalpha-amylases are derived from filamentous fungal strains such asAspergillus, such as A. oryzae and A. niger (e.g. FUNGAMYL and CLARASEL), and Trichoderma, Rhizopus, Mucor, and Penicillium.

Commercially available alpha-amylases contemplated for use in themethods of the invention include; SPEZYME AA; SPEZYME FRED; SPEZYMEETHYL; GZYME G997; CLARASE L (Genencor International Inc.); TERMAMYL120-L, LC, SC and SUPRA (Novozymes Biotech); LIQUOZYME X and SAN SUPER(Novozymes A/S) and ULTRA THIN (/Valley Research).

Plant Enzymes—

Plants have naturally occurring starch degrading enzymes such asalpha-amylases (EC 3.1.1.1); beta-amylases (EC 3.1.1.2),amyloglucosidases (glucoamylase) (EC 3.1.1.3) and starch phosphorylases(EC 2.4.1.1). In addition, plants may have been genetically engineeredto include heterologous genes encoding starch degrading enzymes, such asamylases, glucoamylase and others (WO 03/018766 and WO 05/096804).Endogenous starch degrading plant enzymes, whether naturally occurringor expressed from an introduced polynucleotide, with exposure toelevated temperatures, such as the temperatures of jet cooking or eventemperatures above the gelatinization temperature of granular starchwill become inactivated. However, at temperatures conducted in thepresent process, it is believed that the endogenous starch degradingenzymes are not inactivated and actually contribute to the hydrolysis ofgranular starch. Although not bound to theory, the inventors believethat the alpha-amylase provided in the contacting step modifies thegranular starch structure of the plant material allowing for theproduction of oligosaccharides including dextrins. The oligosaccharidesare further degraded at the temperatures encompassed by the contactingstep (e.g. 45° C. to 70° C.) by plant starch degrading enzymes. Theplant starch degrading enzymes act on the partially hydrolyzed starch toproduce glucose. While exogenous sources of glucoamylases may beincluded in the contacting step, the addition of exogenous glucoamylaseis not required to provide glucose, which is then optionally used as afeedstock for alcohol fermentation. Therefore in one embodiment, thecontacting step of the invention does not include the addition ofglucoamylases derived from microbial sources. However, the addition ofglucoamylases and/or other enzymes such as phytases and proteases mayincrease the production of solubilized granular starch.

Fermenting Organisms—

Examples of fermenting organisms are ethanologenic microorganisms orethanol producing microorganisms such as ethanologenic bacteria whichexpress alcohol dehydrogenase and pyruvate dehydrogenase and which canbe obtained from Zymomonas moblis (See e.g. U.S. Pat. No. 5,000,000;U.S. Pat. No. 5,028,539, U.S. Pat. No. 5,424,202; U.S. Pat. No.5,514,583 and U.S. Pat. No. 5,554,520). In additional embodiments, theethanologenic microorganisms express xylose reductase and xylitoldehydrogenase, enzymes that convert xylose to xylulose. In furtherembodiments, xylose isomerase is used to convert xylose to xylulose. Inparticularly preferred embodiments, a microorganism capable offermenting both pentoses and hexoses to ethanol are utilized. Forexample, in some embodiments the microorganism may be a natural ornon-genetically engineered microorganism or in other embodiments themicroorganism may be a recombinant microorganism. For example, in someembodiments the preferred fermenting microorganisms include bacterialstrains from Bacillus, Lactobacillus, E. coli, Erwinia, Pantoea (e.g.,P. citrea) and Klebsiella (e.g. K. oxytoca). (See e.g. U.S. Pat. No.5,028,539, U.S. Pat. No. 5,424,202 and WO 95/13362).

In further preferred embodiments, the ethanol-producing microorganism isa fungal microorganism, such as a yeast and specifically Saccharomycessuch as strains of S. cerevisiae (U.S. Pat. No. 4,316,956). A variety ofS. cerevisiae are commercially available and these include but are notlimited to FALI (Fleischmann's Yeast), SUPERSTART (Alltech), FERMIOL(DSM Specialties), RED STAR (Lesaffre) and Angel alcohol yeast (AngelYeast Company, China).

Secondary Enzymes—

While in one embodiment, it is contemplated that additional starchhydrolyzing enzymes are not needed, and therefore not included in thecontacting step, additional enzymes may be included in both thecontacting step and fermenting step encompassed by the invention. Insome embodiments, these enzymes will be included as a secondary enzymein the contacting step, which comprises contacting the granular starchslurry with an alpha-amylase and one or more secondary enzymes. In otherembodiments, the additional enzymes will be included in the fermentationstep along with yeast and other components.

In some embodiments during the contacting step with the alpha-amylase,the secondary enzyme may include a glucoamylase, granular starchhydrolyzing enzymes, a protease, a phytase, a cellulase, ahemicellulases, a pullulanase, a xylanase, a lipase, a cutinase, apectinase, a beta-glucanase, a beta amylase, a cyclodextrintransglycosyltransferase and combinations thereof. In some preferredembodiments, the contacting step will include a combination of analpha-amylase, a phytase and optionally a protease. In otherembodiments, the contacting step will include a combination of analpha-amylase, a glucoamylase and optionally a protease. In yet otherembodiments, the contacting step will include a combination of analpha-amylase, a glucoamylase, a phytase and optionally a protease.

Glucoamylases (GA) (E.C. 3.2.1.3.) may be derived from the heterologousor endogenous protein expression of bacteria, plants and fungi sources.Preferred glucoamylases useful in the compositions and methods of theinvention are produced by several strains of filamentous fungi andyeast. In particular, glucoamylases secreted from strains of Aspergillusand Trichoderma are commercially important. Suitable glucoamylasesinclude naturally occurring wild-type glucoamylases as well as variantand genetically engineered mutant glucoamylases. The followingglucoamylases are nonlimiting examples of glucoamylases that may be usedin the process encompassed by the invention. Aspergillus niger G1 and G2glucoamylase (Boel et al., (1984) EMBO J. 3:1097-1102; WO 92/00381, WO00/04136 and U.S. Pat. No. 6,352,851); Aspergillus awamori glucoamylases(WO 84/02921); Aspergillus oryzae glucoamylases (Hata et al., (1991)Agric. Biol. Chem. 55:941-949) and Aspergillus shirousami. (See Chen etal., (1996) Prot. Eng. 9:499-505; Chen et al. (1995) Prot. Eng.8:575-582; and Chen et al., (1994) Biochem J. 302:275-281).

Glucoamylases are also obtained from strains of Talaromyces such asthose derived from T. emersonii, T. leycettanus, T. duponti and T.thermophilus (WO 99/28488; U.S. Pat. No. RE: 32,153; U.S. Pat. No.4,587,215); strains of Trichoderma, such as T. reesei and particularlyglucoamylases having at least 80%, 85%, 90% and 95% sequence identity toSEQ ID NO: 4 disclosed in US Pat. Pub. No. 2006-0094080; strains ofRhizopus, such as R. niveus and R. oryzae; strains of Mucor and strainsof Humicola, such as H. grisea (See, Boel et al., (1984) EMBO J.3:1097-1102; WO 92/00381; WO 00/04136; Chen et al., (1996) Prot. Eng.9:499-505; Taylor et al., (1978) Carbohydrate Res. 61:301-308; U.S. Pat.No. 4,514,496; U.S. Pat. No. 4,092,434; and Jensen et al., (1988) Can.J. Microbiol. 34:218-223). Other glucoamylases useful in the presentinvention include those obtained from Athelia rolfsii and variantsthereof (WO 04/111218).

Enzymes having glucoamylase activity used commercially are produced forexample, from Aspergillus niger (trade name DISTILLASE, OPTIDEX L-400and G ZYME G990 4X from Genencor International Inc.) or Rhizopus species(trade name CU.CONC from Shin Nihon Chemicals, Japan). Also thecommercial digestive enzyme, trade name GLUCZYME from AmanoPharmaceuticals, Japan (Takahashi et al., (1985) J. Biochem.98:663-671). Additional enzymes include three forms of glucoamylase(E.C.3.2.1.3) of a Rhizopus sp., namely “Gluc1” (MW 74,000), “Gluc2” (MW58,600) and “Gluc3” (MW 61,400). Also the enzyme preparation GC480(Genencor International Inc.) finds use in the invention.

Granular starch hydrolyzing enzymes (GSHEs) are able to hydrolyzegranular starch, and these enzymes have been recovered from fungal,bacterial and plant cells such as Bacillus sp., Penicillium sp.,Humicola sp., Trichoderma sp. Aspergillus sp. Mucor sp. and Rhizopus sp.In one embodiment, a particular group of enzymes having GSH activityinclude enzymes having glucoamylase activity and/or alpha-amylaseactivity (See, Tosi et al., (1993) Can. J. Microbiol. 39:846-855). ARhizopus oryzae GSHE has been described in Ashikari et al., (1986)Agric. Biol. Chem. 50:957-964 and U.S. Pat. No. 4,863,864. A Humicolagrisea GSHE has been described in Allison et al., (1992) Curr. Genet.21:225-229; WO 05/052148 and European Patent No. 171218. An Aspergillusawamori var. kawachi GSHE has been described by Hayashida et al., (1989)Agric. Biol. Chem. 53:923-929. An Aspergillus shirousami GSHE has beendescribed by Shibuya et al., (1990) Agric. Biol. Chem. 54:1905-1914.

In one embodiment, a GSHE may have glucoamylase activity and is derivedfrom a strain of Humicola grisea, particularly a strain of Humicolagrisea var. thermoidea (see, U.S. Pat. No. 4,618,579). In some preferredembodiments, the Humicola enzyme having GSH activity will have at least85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity to theamino acid sequence of SEQ ID NO: 3 of WO 05/052148.

In another embodiment, a GSHE may have glucoamylase activity and isderived from a strain of Aspergillus awamori, particularly a strain ofA. awamori var. kawachi. In some preferred embodiments, the A. awamorivar. kawachi enzyme having GSH activity will have at least 85%, 90%,92%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity to the amino acidsequence of SEQ ID NO: 6 of WO 05/052148.

In another embodiment, a GSHE may have glucoamylase activity and isderived from a strain of Rhizopus, such as R. niveus or R. oryzae. Theenzyme derived from the Koji strain R. niveus is sold under the tradename “CU CONC or the enzyme from Rhizopus sold under the trade nameGLUZYME.

Another useful GSHE having glucoamylase activity is SPIRIZYME Plus(Novozymes A/S), which also includes acid fungal amylase activity.

In another embodiment, a GSHE may have alpha-amylase activity and isderived from a strain of Aspergillus such as a strain of A. awamori, A.niger, A. oryzae, or A. kawachi and particularly a strain of A. kawachi.

In some preferred embodiments, the A. kawachi enzyme having GSH activitywill have at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99%sequence identity to the amino acid sequence of SEQ ID NO: 3 of WO05/118800 and WO 05/003311.

In some embodiments, the enzyme having GSH activity is a hybrid enzyme,for example one containing a catalytic domain of an alpha-amylase suchas a catalytic domain of an Aspergillus niger alpha-amylase, anAspergillus oryzae alpha-amylase or an Aspergillus kawachi alpha-amylaseand a starch binding domain of a different fungal alpha-amylase orglucoamylase, such as an Aspergillus kawachi or a Humicola grisea starchbinding domain. In other embodiments, the hybrid enzyme having GSHactivity may include a catalytic domain of a glucoamylase, such as acatalytic domain of an Aspergillus sp., a Talaromyces sp., an Altheasp., a Trichoderma sp. or a Rhizopus sp. and a starch binding domain ofa different glucoamylase or an alpha-amylase. Some hybrid enzymes havingGSH activity are disclosed in WO 05/003311, WO 05/045018; Shibuya etal., (1992) Biosci. Biotech. Biochem 56: 1674-1675 and Cornett et al.,(2003) Protein Engineering 16:521-520.

Suitable proteases include microbial proteases, such as fungal andbacterial proteases, for example, acid fungal proteases such as NSP24and also GC106 (Genencor International Inc.). Preferred fungal proteasesare derived from strains of Aspergillus (e.g. proteases from A. nigerand A. oryzae), Mucor (e.g. M. miehei), Trichoderma, Rhizopus, andCandida. Preferred bacterial proteases are derived from strains ofBacillus such as B. amyloliquefaciens. Proteases added to thefermentation may increase the free amino nitrogen level and increase therate of metabolism of the yeast and further give higher fermentationefficiency.

Enzymes that may be used in the methods of the invention includebeta-amylases (E.C. 3.2.1.2). These are exo-acting maltogenic amylases,which catalyze the hydrolysis of 1,4-alpha-glucosidic linkages inamylose, amylopectin and related glucose polymers. Commercialbeta-amylases are available from Genencor International Inc., andexamples include SPEZYME BBA and OPTIMALT BBA.

Cellulases (E.C. 3.2.1.4) such as endo-glucanases may be used in themethods of the invention. Examples of cellulases include cellulases fromfilamentous fungus such as Trichoderma, Humicola, Fusarium, andAspergillus. Commercially cellulases are available as SPEZYME CP andLAMINEX (Genencor International, Inc) and CELLUZYME and ULTRAFLO(Novozymes A/S).

Xylanases useful in the methods of the invention may be from bacterialor fungal sources, such as Aspergillus, Trichoderma, Neurospora, andFusarium. Commercial preparations include SPEZYME CP and LAMINEX(Genencor International, Inc.) and ULTRAFLO (Novozymes A/S).

A number of bacterial and fungal phytases (E.C. 3.1.3.8 and 3.1.3.26)are known and in some embodiments the addition of phytases areparticularly useful in the methods. Yeast phytases may be derived fromstrains of Saccharomyces (e.g. S. cerevisiae) and Schwanniomyces (e.g.S. occidentalis) (Wodzinski et al., Adv. Apple. Microbiol., 42:263-303).Other fungal phytases have been described in the literature andreference is made to Wyss et al., (1999) Appl. Environ Microbiol.65:367-373; Berka et al., (1998) Appl. Environ. Microbiol. 64:4423-4427; Yamada et al., (1986) Agric. Biol. Chem. 322:1275-1282; PCTPublication Nos. WO 98/28408; WO 98/28409; WO 97/38096 and WO 9844125;and U.S. Pat. Nos. 6,734,004; 6,350,602; and 5,863,533). Fungal phytaseshave been derived from Aspergillus (e.g. A. niger, A. awamori, A.terreus, A. oryzea and A. fumigatus); Thermomyces (Humicola)lanuginousus; Fusarium (F. javanicum and F. versillibodes). Bacterialphytases may also find use in the invention (Greiner R. et al. (1993)Arch. Biochem. Biophys. 303: 107-113; Yoon S. J. et al. (1996) Enzymeand Microbial Technol. 18: 449-454; and WO 06/043178).

Commercially available phytases which may be used according to theinvention include PHYZYME XP 5000 (Danisco A/S); FINASE (Altech); GC491; FINASE, SPEZYME HPA (Genencor), BIO-FEED PHYTASE and PHYTASE NOVO(Novozymes) and NATUPHOS (DSM).

One skilled in the art can readily determine the effective amount of theenzymes which may be used in the process steps encompassed by theinvention.

(B) Process Steps

The granular starch (e.g. milled cereal grain) to be processed is mixedwith an aqueous solution to obtain a slurry. The aqueous solution may beobtained, for example from water, thin stillage and/or backset.

A slurry may have a DS of between 5-60%; 10-50%; 15-45%; 15-30%; 20-45%;20-30% and also 25-40%. The contacting step with an alpha-amylase isconducted at a pH range of 3.5 to 7.0; also at a pH range of 3.5 to 6.5;preferably at a pH range of 4.0 to 6.0 and more preferably at a pH rangeof 4.5 to 5.5. The slurry is held in contact with the alpha-amylase at atemperature below the starch gelatinization temperature of the granularstarch. In some embodiments, this temperature is held between 45° C. and70° C.; in other embodiments, the temperature is held between 50° C. and70° C.; between 55° C. and 70° C.; between 60° C. and 70° C., between60° C. and 65° C.; between 55° C. and 65° C. and between 55° C. and 68°C. In further embodiments, the temperature is at least 45° C., 48° C.,50° C., 53° C., 55° C., 58° C., 60° C., 63° C., 65° C. and 68° C. Inother embodiments, the temperature is not greater than 65° C., 68° C.,70° C., 73° C., 75° C. and 80° C.

The initial starch gelatinization temperature ranges for a number ofgranular starches which may be used in accordance with the processesherein include barley (52° C. to 59° C.), wheat (58° C. to 64° C.), rye(57° C. to 70° C.), corn (62° C. to 72° C.), high amylose corn (67° C.to 80° C.), rice (68° C. to 77° C.), sorghum (68° C. to 77° C.), potato(58° C. to 68° C.), tapioca (59° C. to 69° C.) and sweet potato (58° C.to 72° C.). (J. J. M. Swinkels pg 32-38 in STARCH CONVERSION TECHNOLOGY,Eds Van Beynum et al., (1985) Marcel Dekker Inc. New York and TheAlcohol Textbook 3^(rd) ED. A Reference for the Beverage, Fuel andIndustrial Alcohol Industries, Eds Jacques et al., (1999) NottinghamUniversity Press, UK).

In the contacting step, the slurry may be held in contact with thealpha-amylase for a period of 5 minutes to 48 hours; and also for aperiod of 5 minutes to 24 hours. In some embodiments the period of timeis between 15 minutes and 12 hours, 15 minutes and 6 hours, 15 minutesand 4 hours and also 30 minutes and 2 hours.

The effective concentration of the alpha-amylase used in the contactingstep will vary according to the specific process conditions and granularstarch used. However, in general the amount of alpha-amylase used willbe in the range of 0.001 to 50 AAU/g DS, 0.01 to 30 AAU/g DS, 0.01 to 10AAU/g DS and also 0.05 to 5.0 AAU/g DS.

In some embodiments, the effective dose of an alpha-amylase in thecontacting step and/or fermentation step will be 0.01 to 15 SSU/g DS;also 0.05 to 10 SSU/g DS; also 0.1 to 10 SSU/g DS; and 0.5 to 5 SSU/gDS.

In some embodiments, the effective dose of a glucoamylase for thecontacting step and/or the fermentation step will be in the range of0.01 to 15 GAU/g DS; also 0.05 to 10 GAU/g DS; also 0.1 to 10 GAU/g DSand even 0.5 to 5 GAU/g DS.

In some embodiments, the effective dose of a phytase to be used in thecontacting step and/or fermentation step will be in the range of 0.001to 15 FTU/g DS; also 0.005 to 10 FTU/g DS; and also 0.05 to 5 FTU/g DS.One phytase unit (FTU) is the amount of enzyme, which liberates 1micromole inorganic phosphorus per minute from sodium phytate, 0.0051moles/liter, at 37° C. and at pH 5.0.

In some embodiments, the effective dose of a protease to be used in thecontacting step and/or fermentation step will be in the range of 0.01 to15 SAPU/g DS; also 0.01 to 10 SAPU/g DS; and also 0.05 to 5 SAPU/g DS.SAPU refers a spectrophotometric acid protease unit, wherein 1 SAPU isthe amount of protease enzyme activity that liberates one micromole oftyrosine per minute from a casein substrate under conditions of theassay.

During the contacting step between 25-90% of the granular starch issolubilized to produce oligosaccharides comprising dextrin. In someembodiments, greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85% and 90% of the granular starch is solubilized.

After contacting the granular starch with the alpha-amylase for a periodof time as indicated above, a soluble starch substrate (mash) isobtained which comprises greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75% and 80% glucose.

In some preferred embodiments of the contacting step, a slurrycomprising granular corn starch having a DS of 20-40% is contacted withan alpha-amylase derived from Bacillus stearothermophilus or Bacilluslicheniformis for 1 to 6 hours at a temperature between 55° C. to 70° C.to obtain a soluble starch substrate comprising at least 30% glucose. Inother preferred embodiments of the contacting step, a slurry comprisinggranular milo starch having a DS of 20-40% is contacted with analpha-amylase derived from Bacillus stearothermophilus or Bacilluslicheniformis for 1 to 6 hours at a temperature between 55° C. to 70° C.to obtain a soluble starch substrate comprising at least 50% glucose.

After the contacting step which results in the production of a mashcomprising glucose, the mash is subjected to fermentation with afermenting microorganism (e.g. an ethanol-producing microorganism).

However, prior to subjecting the mash including at least 10% glucose tofermentation, the mash may be further exposed to an aqueous solutioncomprising, for example backset and/or corn steep and adjusted to a pHin the range of pH 3.0 to 6.0; pH 3.5 to 5.5, or pH 4.0 to 5.5. In thisembodiment of the invention, the % DS of the mash may be diluted. Forexample, the DS of the diluted mash maybe between 5 to 35%; 5 to 30%; 5to 25%; 5 to 20%; 5 to 20%; 5 to 15%; and 5 to 10% less than the % DS ofthe slurry in the contacting step. In one non-limiting example, if the %DS of the slurry in the contacting step is approximately 32% and themash is further exposed to a diluting aqueous solution which dilutes theDS between 5 to 10%, the DS of the mash to be fermented will be between22% and 27%. In some preferred embodiments, if the DS of the contactingslurry is between 30 to 35%, the DS of the diluted slurry will be about20 to 30%.

In a preferred embodiment, the mash comprising at least 10% glucose isthen subjected to fermentation processes using fermenting microorganismsas described above. These fermentation processes are described in TheAlcohol Textbook PED, A Reference for the Beverage, Fuel and IndustrialAlcohol Industries, Eds Jacques et al., (1999) Nottingham UniversityPress, UK.

In some preferred embodiments, the mash is fermented with a yeast attemperatures in the range of 15 to 40° C. and also 25 to 35° C.; at a pHrange of pH 3.0 to 6.5; also pH 3.0 to 6.0; pH 3.0 to 5.5, pH 3.5 to 5.0and also pH 3.5 to 4.5 for a period of time of 12 to 240 hours,preferably 12 to 120 and more preferably from 24 to 90 hours to producean alcohol product, preferably ethanol.

Yeast cells are generally supplied in amounts of 10⁴ to 10¹², andpreferably from 10⁷ to 10¹⁰ viable yeast count per ml of fermentationbroth. The fermentation will include in addition to a fermentingmicroorganisms (e.g. yeast) nutrients, optionally acid and additionalenzymes.

In one preferred embodiment, the contacting step is conducted in aseparate vessel from the fermenting step. It is also contemplated thatthe contacting step and fermenting step may be conducted in a SSFprocess in the same vessel.

In some embodiments, in addition to the raw materials described above,fermentation media will contain supplements including but not limited tovitamins (e.g. biotin, folic acid, nicotinic acid, riboflavin),cofactors, and macro and micro-nutrients and salts (e.g. (NH4)₂SO₄;K₂HPO₄; NaCl; MgSO₄; H₃BO₃; ZnCl₂; and CaCl₂).

Additional enzymes to be included in the fermentation step may be thesame or different from the enzymes used in the contacting step. In someembodiments, the enzyme will include alpha-amylases and glucoamylases,including granular starch hydrolyzing enzymes. In some preferredembodiments, the glucoamylase and alpha-amylase may occur in a blend.Particularly preferred enzyme blends include STARGEN 001 (GenencorInternational Inc.), which is a blend of an alpha-amylase from A.kawachi and a glucoamylase from A. niger. In some preferred embodiments,the glucoamylase will be derived from a Trichoderma reesei glucoamylase,a Athelia rolfi glucoamylase, a Talaromyces glucoamylase, a Aspergillusglucoamylase and hybrid and variants glucoamylase derived there from. Insome preferred embodiments, the enzyme is selected from a cellulase, aphytase and a protease.

Recovery of Alcohol and Other End Products—

The preferred end product of the instant fermentation process is analcohol product, preferably ethanol. The end product produced accordingto the process may be separated and/or purified from the fermentationmedia. Methods for separation and purification are known, for example bysubjecting the media to extraction, distillation and columnchromatography. In some embodiments, the end product is identifieddirectly by submitting the media to high-pressure liquid chromatography(HPLC) analysis.

In further embodiments, the mash may be separated by for examplecentrifugation into the liquid phase and solids phase and end productssuch as alcohol and solids recovered. The alcohol may be recovered bymeans such as distillation and molecular sieve dehydration or ultrafiltration.

In some embodiments, the yield of ethanol will be greater than 8%, 10%,12%, 14%, 16% and 18% by volume. The ethanol obtained according toprocess of the invention may be used as a fuel ethanol, potable ethanolor industrial ethanol.

In further embodiments, the end product may include the fermentationco-products such as distillers dried grains (DDG) and distiller's driedgrain plus solubles (DDGS), which may be used as an animal feed.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.Indeed, it is contemplated that these teachings will find use in furtheroptimizing the process systems described herein.

In the disclosure and experimental section which follows, the followingabbreviations apply: GA (glucoamylase); wt % (weight percent); ° C.(degrees Centigrade); H₂O (water); dH₂O (deionized water); dIH₂O(deionized water, Milli-Q filtration); g or gm (grams); μg (micrograms);mg (milligrams); kg (kilograms); μL (microliters); ml and mL(milliliters); mm (millimeters); μm (micrometer); M (molar); mM(millimolar); μM (micromolar); U (units); MW (molecular weight); sec(seconds); min(s) (minute/minutes); hr(s) (hour/hours); DO (dissolvedoxygen); W/V (weight to volume); W/W (weight to weight); V/V (volume tovolume); Genencor (Genencor International, Inc., Palo Alto, Calif.); MT(Metric ton); and ETOH (ethanol).

The Following Enzyme Preparations were Used in the Examples Below:

SPEZYME Ethyl (available from Genencor)—a bacterial alpha-amylaseobtained from a genetically modified strain of Bacillus licheniformis.

GC100 —an experimental bacterial alpha-amylase disclosed in US2006/0014265.

Humicola grisea glucoamylase (HGA) having the amino acid sequencedisclosed as SEQ ID NO: 3 of WO 2005/052148.

STARGEN 001 (available from Genencor)—a blend of Aspergillus nigerglucoamylase and Aspergillus kawachi alpha-amylase.

The Following Assays were Used in the Examples Below:

The activity of alpha-amylase is expressed as alpha amylase units (AAU)and enzyme activity was determined by the rate of starch hydrolysis, asreflected in the rate of decrease of iodine-staining capacity, which wasmeasured spectrophotometrically. One AAU of bacterial alpha-amylaseactivity is the amount of enzyme required to hydrolyze 10 mg of starchper min under standardized conditions.

Alpha-amylase activity made also determined as soluble starch unit (SSU)and is based on the degree of hydrolysis of soluble potato starchsubstrate (4% DS) by an aliquot of the enzyme sample at pH 4.5, 50° C.The reducing sugar content is measured using the DNS method as describedin Miller, G. L. (1959) Anal. Chem. 31:426-428. One unit of the enzymeactivity (SSU) is equivalent to the reducing power of 1 mg of glucosereleased per minute at the specific incubation conditions.

Glucoamylase activity was measured using a well-known assay which isbased on the ability of glucoamylase to catalyze the hydrolysis ofp-nitrophenyl-alpha-D-glucopyranoside (PNPG) to glucose andp-nitrophenol. At an alkaline pH, the nitrophenol; forms a yellow colorthat is proportional to glucoamylase activity and is monitored at 400 nmand compared against an enzyme standard measured as a GAU.

One “Glucoamylase Activity Unit” (GAU) is the amount of enzyme that willproduce 1 gm of reducing sugar, calculated as glucose per hour from asoluble starch substrate (4% DS) at pH 4.2 and 60° C.

Brix, the measurement of total solublizied solid content at a giventemperature was determined by measurement with a Refractometer.

Determination of total starch content: The enzyme-enzyme starchliquefaction and saccharification process was used to determine thetotal starch content. In a typical analysis, 2 g of dry sample was takenin a 100 ml Kohlraucsh flask and 45 ml of MOPS buffer, pH 7.0 was added.The slurry was well stirred for 30 min. SPEZYME FRED (1:50 diluted inwater) (Genencor), 1.0 ml was added and heated to boiling for 3-5 min.The flask was placed in an autoclave maintained at 121° C. for 15 min.After autoclaving the flask was placed in a water bath at 95° C. and 1ml of 1:50 diluted SPEZYME FRED was added and incubated for 45 min. ThepH was adjusted to pH 4.2 and the temperature was reduced to 60° C. Thiswas followed by addition of 20 ml acetate buffer, pH 4.2.Saccharification was carried out by adding 1.0 ml of 1:100 dilutedOPTIDEX L-400 (Genencor) and the incubation was continued for 18 hr at60° C. The enzyme reaction was terminated by heating at 95° C. for 10min. The total sugar composition was determined by HPLC analysis usingglucose as a standard. The soluble starch hydrolysate from waterextraction of a sample at room temperature without enzymatic treatmentwas subtracted from the total sugar.

Determination of the % solubilized solids—a 7 ml sample was placed in asmall screw cap test tube (pH adjusted to 5.0 to 6.0) and 0.007 mlSPEZYME Fred was added to the tube. The test tube was placed in aboiling water bath for 10 min and gently mixed at various times duringthe incubation. After 10 min the tube was removed and placed in a 80° C.water bath for 1 hr. The tube was cooled and centrifuges. The Brix ofthe supernatant was determined and compared to a control sample. The %solubilized solids=control Brix×100/sample Brix.

Ethanol and carbohydrate determinations of the samples were determinedusing the HPLC method as follows:

a 1.5 mL Eppendorf centrifuge tube was filled with fermentor mash andcooled on ice for 10 min; the sample tube was centrifuged for 1 min inan Eppendorf table top centrifuge; a 0.5 mL sample of the supernatantwas transferred to a test tube containing 0.05 mL of 1.1N H₂SO₄ andallowed to stand for 5 min; 5.0 mL of water was added to the test tubeand then the sample was filtered into a HPLC vial through 0.2 μm NylonSyringe Filter; and run on HPLC. The HPLC conditions included:

Ethanol System: Column: Phenomenex Rezex Organic Acid Column(RHM-Monosaccharide) #00H 0132-KO (Equivalent to Bio-Rad 87H); ColumnTemperature: 60° C.; Mobile Phase: 0.01 N H₂SO₄; Flow Rate: 0.6 mL/min;Detector: RI; and Injection Volume: 20 μL.

Carbohydrate System: Column: Phenomenex Rezex Carbohydrate(RCM-Monosaccharide) #00H-0130-KO (Equivalent to Bio-Rad 87H); ColumnTemperature: 70° C.; Mobile Phase: Nanopure DI H₂O; Flow Rate: 0.8mL/min; Detector: RI; Injection Volume: 10 μL (3% DS material).

The column separated based on the molecular weight of the saccharides,which are designated as DP1 (glucose); DP2 (disaccharides); DP3(trisaccharides) and DP>3 (oligosaccharide sugars having a degree ofpolymerization greater than 3).

Example 1 Solubilization and Ethanol Production from Granular Starch ofWhole Ground Corn and Fractionated Corn

This experiment was run on three different corn granular starchsubstrates (A) 370 g of whole ground corn having a moisture content of13.3%; (B) 354.2 g corn endosperm having a moisture content of 9.2%; and(C) refined corn starch obtained from having a moisture content of11.8%. Each substrate was weighed and transferred to a stainless steelvessel to make a final 1000 g slurry with water corresponding to 32% DS.

The pH of the slurry was adjusted to pH 5.5 using 6N H₂SO₄. GC100 (4.0AAU/g DS) was added. The temperature was maintained at 60° C. During theincubation the slurry was gently stirred with an overhead mixer. Aftertime internals of 2, 4, 6, 12 and 24 hours, the BRIX, % solubilzedstarch and sugar compositions (% W/W) were determined, and the resultsare illustrated in Table 1. At 24 hours, 79.1%, 71.1% and 60.0% of thegranular starch from whole ground corn, endosperm and refined sugar wassolubilized during the contacting step respectively. The % glucose ofthe hydrolyzate at 24 hours was 65.22% for whole ground corn, 49.64% forendosperm and only 5.79% for refined starch.

TABLE 1 % Starch % % % Grain Time BRIX Solubilized Glucose DP2 DP3 DP >3 Whole 2 11.5 31.60 21.36 13.48 33.56 corn 4 14.0 37.56 24.31 13.6524.48 6 16.3 41.33 25.42 13.36 19.88 12 18.2 46.23 26.18 12.63 14.96 2420.0 79.1 65.22 22.50 7.36 4.92 Endo- 2 13.4 27.12 13.85 8.88 50.15sperm 4 16.1 33.01 16.45 9.39 41.15 6 17.5 36.56 18.18 9.59 35.67 1221.7 47.43 20.98 9.65 21.94 24 22.9 71.1 49.64 21.33 9.52 19.51 Refined2 14.4 1.40 10.37 15.27 72.96 Cornstarch 4 16.8 2.34 11.60 15.22 70.84 617.8 5.14 12.27 15.13 67.45 12 19.1 4.94 13.21 15.62 66.22 24 20.9 60.05.79 14.40 17.42 62.39

After 24 hours, using the samples from whole ground corn (32% DS), yeastfermentations were conducted at pH 4.2, 32° C. in the presence of 400ppm urea; Red Star Red yeast (Fermentus); STARGEN 001; and 0.1 SAPU/g DSprotease in a 125 ml flask. HPLC data are illustrated in Table 2.

TABLE 2 STARGEN 001 % V/V ETOH % V/V ETOH % V/V ETOH GAU/g 24 hrs 48 hrs72 hrs 0.1 10.11 11.74 13.64 0.2 10.36 12.19 14.62 0.4 10.83 12.73 15.35

Example 2 Solubilization and Ethanol Production from Milo GranularStarch

Two pretreatments were run: (A) 160 g of whole ground milo having amoisture content of 11.6% and a total starch content of 53.3% wasweighed and transferred to a stainless steel vessel containing 340 gwater. The pH of the slurry was adjusted to pH 5.5 using 6 N sulphuricacid. SPEZYME Ethyl (1.0 AAU/g DS) was added. The temperature wasmaintained at 62° C. and 32% DS. (B) HGA was included in thepretreatment as described above in (A) at the equivalent of 0.1 GAUHGA/g DS. The % solubilized solids and % glucose are presented in Table3.

TABLE 3 % Time % DP1 % solublized Enzyme (hrs) (glucose) % DP2 % DP3DP > 3 starch SPEZYME 2 57.71 24.60 10.42 7.27 Ethyl 4 64.41 22.50 9.133.95 6 67.16 21.83 8.16 2.85 24 86.50 9.91 2.95 0.63 54.7 SPEZYME 284.34 9.48 1.47 4.72 Ethyl + 4 87.72 8.07 1.18 3.03 HGA 6 88.91 7.681.03 2.37 24 93.10 5.26 0.59 1.05 69.3

The feedstock (mash) from the HGA pretreatment described above wasevaluated under regular yeast fermentation conditions (e.g. Red StarYeast, pH 4.2, 32° C. in the presence of 400 ppm urea; STARGEN 001 and0.05 SAPU/g DS) in a 125 ml flask. HPLC results are illustrated in Table4.

TABLE 4 GAU/g % V/V Ethanol % V/V Ethanol Pre-treatment STARGEN 001 24hrs 48 hrs SPEZYME 0.1 10.02 11.98 Ethyl + HGA 0.2 10.22 12.75

Example 3 Effect of Temperature on Glucose Production from Whole GroundMilo

Incubation of a 30% ds aqueous slurry of whole ground milo at pH 5.5containing GC100 (4.0 AAU/g DS) was carried out at 60° C., 65° C. and70° C. After 6 hours of the incubation, the samples were withdrawn andcentrifuged to separate the insolubles. The Brix and HPLC composition ofthe clear supernatant was measured. The % solubilized starch and %glucose were determined and the results are illustrated in Table 5.

TABLE 5 % % W/W Solubilized DP1 % W/W % W/W % W/W ° C. Starch (Glucose)DP2 DP3 DP > 3 60 69.9 52.48 27.07 12.08 8.36 65 68.2 52.61 23.42 10.4113.56 70 61.9 36.49 18.98 10.27 34.26

As the incubation temperature increased from 60° C. to 70° C. during thecontacting step with whole ground milo, the solubilization of starch andthe glucose content decreased. This suggests that the alpha-amylase maybe inactivated at 70° C. More than 50% of the solubilized starch washydrolyzed to glucose at 65° C. suggesting the endogenous plant starchhydrolyzing enzymes are capable of hydrolyzing the solubleoligosaccharides into glucose.

The feedstock from the pretreatment described above at 65° C. wasevaluated under regular fermentation conditions as essentially describedin example 2; except the % DS was 30. The results are illustrated inTable 6.

TABLE 6 STARGEN 001 % V/V ETOH % V/V ETOH % V/V ETOH (GAU/g DS) 24 hrs48 hrs 72 hrs 0.1 9.19 11.00 11.29 0.2 9.32 11.07 12.80 0.4 9.61 11.4713.32

Example 4 Solubilization and Ethanol Production from Rice GranularStarch

Rice grain (116 g) having a starch content of 81.5%; a moisture contentof 14% and a particle size that passes through a 30 mesh screen wasmixed with 284 g of water to make a 25% DS slurry. GC100 (4.0 AAU/g DS)was added to the slurry. The temperature was maintained at 65° C. and pHadjusted to pH 5.5. The Brix was measured at 2, 4 6, and 24 hrs. The %soluble starch and % glucose were determined and the results areillustrated in Table 7.

TABLE 7 % % W/V Time solubilized DP1 % W/V % W/V % W/V hrs Brix starch(glucose) DP2 DP3 .DP3 1 11 39.2 27.5 19.8 12.7 39.6 2 12.5 44.6 31.222.2 13.4 32.6 4 14.8 52.8 33.4 24.0 13.8 28.7 6 16.2 57.5 33.8 24.413.9 27.7 24 16.4 58.4 36 26.2 14.6 23.2

After 24 hours, yeast fermentations were conducted at pH 4.2, 30° C. inthe presence of 0.75 GAU/g DS STARGEN 001, 400 ppm urea, and Angel yeast(Jiangxi, China) at 0.4%. HPLC samples were taken at 24, 48 and 67 hrs(Table 8).

TABLE 8 Time % W/V % V/V hrs glucose ETOH 24 2.15 6.62 48 0.32 10.4 670.35 12.0

1-31. (canceled)
 32. A method of producing a mash comprising contactinga slurry, comprising granular starch obtained by dry milling whole grainwithout removing the germ and bran fractions, with an α-amylase at atemperature below the starch gelatinization temperature of the granularstarch to produce a mash comprising at least 30% (w/w) of the solublestarch substrate in the form of glucose, wherein the contacting isconducted for a period of 5 minutes to 48 hours at a pH of 3.5 to 7.0.33. The method according to claim 32, wherein the contacting isconducted for a period of 2 to 24 hours.
 34. The method according toclaim 32, wherein the temperature is 45° C. to 70° C.
 35. The methodaccording to claim 32, wherein the contacting step is conducted at a pHof between 4.0 and 6.0.
 36. The method according to claim 32, whereinthe slurry has 20% to 45% dry solids (DS) granular starch.
 37. Themethod according to claim 32, wherein the slurry further comprises thinstillage and/or backset.
 38. The method according to claim 32, whereinthe mash comprises at least 50% (w/w) of the soluble starch substrate inthe form of glucose.
 39. The process according to claim 38, wherein themash comprises at least 50% (w/w) of the soluble starch substrate in theform of glucose.
 40. The method according to claim 32, wherein the wholegrain is from corn, wheat, rye, barley, sorghum, or a combinationthereof.
 41. The method according to claim 32, wherein the contactingdoes not involve the addition of another starch hydrolyzing enzyme. 42.The method according to claim 32, further comprising adding aglucoamylase, phytase, protease, β-amylase, cellulase, or hemicellulaseto the contacting step.
 43. The method according to claim 42, whereinthe additional enzyme is a glucoamylase.
 44. The method according toclaim 42, wherein the additional enzyme is a phytase.
 45. The methodaccording to claim 42, wherein the additional enzyme is a protease. 46.The method according to claim 42, wherein the additional enzyme is aβ-amylase.
 47. The method according to claim 32, further comprisingfermenting the mash in a separate reaction vessel in the presence of afermenting microorganism and a starch hydrolyzing enzyme at atemperature of between 10° C. and 40° C. for a period of time of 10hours to 250 hours to produce an end product.
 48. The method accordingto claim 47, further comprising recovering the end product.
 49. Themethod according to claim 48, wherein the end product is an alcohol. 50.The method according to claim 47, further comprising recovering afermentation co-product.